LOCUS       sequence-146361-        4068 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  fission yeast epitope tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4068)
  AUTHORS   Amelina H, Moiseeva V, Collopy LC, Pearson SR, Armstrong CA, Tomita 
            K
  TITLE     Sequential and counter-selectable cassettes for fission yeast.
  JOURNAL   BMC Biotechnol. 2016 Nov 8;16(1):76.
  PUBMED    27825338
REFERENCE   2  (bases 1 to 4068)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "BMC 
            Biotechnol."; date: "2016-11-8"; volume: "16(1)"; pages: "76"
COMMENT     Sequence Label: pNX3c2-FL3
FEATURES             Location/Qualifiers
     source          1..4068
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     43..61
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(99..121)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     221..240
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        321..339
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     321..338
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             412..435
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             439..486
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     terminator      516..703
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(571..595)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     enhancer        728..1031
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1032..1235
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1186..1206
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1280..1299
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1280..1298
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1314..1886
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      1892..2089
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2148..2165)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2319..2907)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2399..2418)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3078..3869,3870..3938))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3701..3720
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3939..4043)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
       61 ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
      121 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
      181 tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta
      241 ctgagagtgc accatatgga catattgtcg ttagaacgcg gctacaatta atacataacc
      301 ttatgtatca tacacatacg atttaggtga cactatagaa cgcggccgcc agctgaagct
      361 tcgtacgctg caggtcgacg gatccccggg ttaattaacg ctagcatggg tgactacaag
      421 gacgacgatg acaagggaga ttacaaagat gacgacgata aggactacaa ggacgacgat
      481 gacaagtcta gatgaggcgc gccacttcta aataagcgaa tttcttatga tttatgattt
      541 ttattattaa ataagttata aaaaaaataa gtgtatacaa attttaaagt gactcttagg
      601 ttttaaaacg aaaattctta ttcttgagta actctttcct gtaggtcagg ttgctttctc
      661 aggtatagta tgaggtcgct cttattgacc acacctctac cggcagatcc gctaggagat
      721 ctcgcgacgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
      781 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
      841 gacgtcaatg ggtggactat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
      901 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
      961 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
     1021 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
     1081 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
     1141 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
     1201 ggcgtgtacg gtgggaggtc tatataagca gagctctctg gctaactaga gaacccactg
     1261 cttactggct tatcgaaatt aatacgactc actataggga gacccaagct tacatgggta
     1321 ccactcttga cgacacggct taccggtacc gcaccagtgt cccgggggac gccgaggcca
     1381 tcgaggcact ggatgggtcc ttcaccaccg acaccgtctt ccgcgtcacc gccaccgggg
     1441 acggcttcac cctgcgggag gtgccggtgg acccgcccct gaccaaggtg ttccccgacg
     1501 acgaatcgga cgacgaatcg gacgacgggg aggacggcga cccggactcc cggacgttcg
     1561 tcgcgtacgg ggacgacggc gacctggcgg gcttcgtggt cgtctcgtac tccggctgga
     1621 accgccggct gaccgtcgag gacatcgagg tcgccccgga gcaccggggg cacggggtcg
     1681 ggcgcgcgtt gatggggctc gcgacggagt tcgcccgcga gcggggcgcc gggcacctct
     1741 ggctggaggt caccaacgtc aacgcaccgg cgatccacgc gtaccggcgg atggggttca
     1801 ccctctgcgg cctggacacc gccctgtacg acggcaccgc ctcggacggc gagcaggcgc
     1861 tctacatgag catgccctgc ccctaatcag tactgacaat aaaaagattc ttgttttcaa
     1921 gaacttgtca tttgtatagt ttttttatat tgtagttgtt ctattttaat caaatgttag
     1981 cgtgatttat attttttttc gcctcgacat catctgccca gatgcgaagt taagtgcgca
     2041 gaaagtaata tcatgcgtca atcgtatgtg aatgctggtc gctatactgc tgtcgattcg
     2101 atactaacgc cgccatccag tttaaacgag ctcgaattct aggcttcctc gctcactgac
     2161 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     2221 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     2281 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     2341 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     2401 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     2461 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca
     2521 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     2581 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     2641 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     2701 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     2761 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     2821 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     2881 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     2941 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     3001 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     3061 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     3121 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     3181 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     3241 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     3301 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     3361 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     3421 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     3481 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     3541 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     3601 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     3661 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     3721 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     3781 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     3841 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     3901 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     3961 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     4021 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacct
//
